Abstract: The objective of this study was to validate NAM model, an artificial mouth system for use in the study of oral biofilms. The NAM model consists of a cylindrical glass chamber (1x6 cm) which was used to mimic the oral cavity and glass beads (3 mm), placed along its length to provide surfaces for biofilm formation. The opening at both ends of the chamber which were fitted with rubber tubing served as an inlet to and outlet from the system. The two tubing were then connected to a reservoir (bacterial reservoir) via a peristaltic pump. The apparatus was kept at 37°C in a water bath. In the formation of oral biofilm, saliva was first pumped into the system to coat the glass beads. Excess saliva was then rinsed with distilled deionised water. Bacterial inoculum (Strep. mutans) was then allowed to flow into the system for 24 h. The bacterial population (cfu mL-1) in the biofilm developed on each of the glass beads in different experiments were analyzed and, validated for reproducibility. Its efficiency in maintaining temperature and flow rate for the experiment and sterility prior to the experiment was also determined. The results obtained showed that the bacterial counts of the biofilms between glass beads are not significantly different (p>0.6) and demonstrated reproducibility (4-5% standard deviation) between different experiments. It was also observed that the flow rate and temperature are constant and sterility of the apparatus is maintained throughout the experiment. This shows that the NAM model is valid for use in the in vitro study of oral biofilm development.
INTRODUCTION
Since the late 19th century, various artificial mouth models had been invented by researchers around the world (Tang et al., 2003). The curiosity to know and understand what is happening in the mouth has led to the development of artificial mouth models at the laboratory scale. The investigations on oral biofilm are made easier with artificial mouth models which allow for the development of biofilm to be monitored easily by using modern microscopy tools or by analyzing data gathered through laboratory trials. A model which can be used to monitor, observe and study the development of biofilm in an environment that mimics the oral cavity has been designed by Tang et al. (2003).
In the early days, the artificial mouth model was designed using glass cylindrical funnel (Pigman et al., 1952; Pigman and Newbrun, 1962). This was later replaced by glass incubation chamber (Russell and Coulter, 1975). Subsequently, artificial mouth models called the Column System using pyrex glass tube (Sudo, 1977) and a culture vessel were designed (De Jong and Van Der Hoeven, 1987).
From 1990 onward, a complex artificial mouth model was developed. The model was called A Multi-Plaque Artificial Mouth, consisting of a glass cylinder containing five stations for plaque growth. This complex artificial mouth model can be used to monitor long-term growth of plaques (Sissons et al., 1991, 1992; Shu et al., 2000; Filoche et al., 2004). An artificial mouth model reported by Busscher et al. (1992) was the Parallel-plate Flow Chamber. The model consists of glass-bottomed plate with connecting flasks.
Later, artificial mouth models using Constant Depth Film Fermentor (CDFF) were developed (Kinniment et al., 1996; Pratten et al., 1998; Pratten and Wilson, 1999; Pratten et al., 2000; Hope and Wilson, 2003; Steinberg et al., 2005; Lamfon et al., 2005). The basis of the CDFF is a rotating turntable containing recessed cavities into which an appropriate substratum can be inserted. Some researchers subsequently used flow cells/flow chamber as artificial mouth models (Weiger et al., 1999; Palmer et al., 2001; Decker et al., 2003; Foster and Kolenbrander, 2004). The flow cells/flow chamber usually consisted of glass slides, glass cover slips or enamel slides as substratum for biofilm formation. An artificial mouth model consisting of silicone rubber tubing packed with glass beads and connected to a peristaltic pump has been designed by Black et al. (2004).
In all of those models, various substrata that include hydroxyapatite beads, cover slips, glass beads and enamel are used for biofilm formation. Peristaltic pumps were used to supply the saliva and nutrients to the developing biofilm. There are attempts by some researchers (Filoche et al., 2005; Takarada et al., 2004; Park et al., 2003; Fathilah and Rahim, 2003) to study biofilm formation by evaluating the bacterial adherence to glass surfaces under planktonic state. Microbes in oral biofilm however, may exhibit different properties from those in planktonic state (Black et al., 2004; Ceri et al., 1999).
The in vivo investigation of oral biofilm development is made difficult by its heterogeneity, limited access, variability and uncontrollable oral environments and ethical problems (Wong and Sissons, 2001). This triggers the interest to design an artificial mouth model to be referred as Nordini`s Artificial Mouth (NAM) model for the development of oral biofilm. The reliability of the model was evaluated for use in the development of biofilm. Thus the main objective of this study was to determine the reproducibility of the bacterial populations formed in the biofilm between glass beads in one experiment and between different experiments. The efficiency of the model in maintaining conditions that mimics the oral environment was also validated.
MATERIALS AND METHODS
Glass Pasteur Pipette and Glass Beads Were Purchased from Merck, Germany
Bacterial reservoir and water bath were custom made and designed with
dimensions suitable for the minimum space in the laboratory. Both were
made from pyrex glass and consist of two parts (top and bottom) with a
total height of 10 cm. The diameter of the water bath is 8.0 cm, whereas
that of the bacterial reservoir, 6.5 cm. The top part of the water bath
has a hole which can be used to fit in a thermometer while its bottom
part has two opposite apertures. For the bacterial reservoir, the top
part is provided with two openings which are opposite to one another.
Brain Heart Infusion agar and Brain Heart Infusion broth were purchased from Oxoid Ltd., Hampshire, England Bacterial strains (Strep. mutans ATTC 25175) were purchased from ATTC, United States (US) in freeze-dried form.
Washing Procedures for the Apparatus and Glass Wares
All washables apparatus and glass wares were soaked overnight in a
diluted solution of Decon 90. The glass wares were then sonicated in an
Ultrasonicator for a few minutes followed by scrubbing with suitable brushes.
The apparatus were rinsed thoroughly with tap water and re-rinsed with
distilled water. The apparatus were then air-dried or oven-dried prior
to sterilization.
Sterilisation and Aseptic Techniques
In this study, heat sterilization methods were employed. Moist heat-sterilisation
was carried out by autoclaving at 121°C at 15 psi for 20 min. All
microbiological media, solutions, glass wares, apparatus and other items
that can withstand high temperature and pressure were sterilized using
this method. Dry heat sterilization method, i.e., direct flaming was used
to sterile the inoculating loops and hockey stick. All procedures, methods
and practices that require aseptic techniques were carried out according
to the method described by Benson (2002).
Setting up of the Nordini`s Artificial Mouth (NAM) Model
A glass pasteur pipette was cut using a diamond blade to give a cylindrical
glass chamber (1x6 cm) to represent the oral cavity. Six glass beads (3
mm in diameter) were placed along the length of the chamber and served
as substrata for biofilm formation. The open-ends of the glass chamber
were connected with rubber tubing to a bacterial reservoir via a peristaltic
pump. The chamber was immersed in a water bath which functions as an incubator,
with temperature fixed at 37°C to mimic the in vivo temperature
of the mouth. A thermometer was fitted into the top part of the incubator
to monitor the temperature of the system. This was to ensure that the
biofilm develops in an environment that mimics that of the mouth. The
peristaltic pump was used to maintain the flow rate that represents the
flow rate of saliva in the oral cavity, of fluids coming from and fluids
flowing back to the bacterial reservoir after passing through the NAM
model.
Preparation of Sterile Saliva
Undiluted sterile saliva was prepared by a slight modification of
the method described by De Jong and Van der Hoeven (1987). About 25 mL
of Stimulated Whole Saliva (SWS) was collected everyday from a single
volunteer. The volunteer was asked to chew on a sugar-free gum to stimulate
saliva production. The collection of SWS was done using ice-chilled test
tubes. The saliva was collected from a single volunteer to minimize any
variations that may arise between individuals. The aggregation of protein
in the SWS samples was minimized by adding 1,4-Dithio-D,L-threitol (DTT)
to a concentration of 2.5 mM. Upon addition of DTT, the saliva was stirred
slowly for 10 min before it was centrifuged at 864 x g for 30 min. The
supernatant obtained was then filter-sterilized through a disposable 0.2
μm (Supor Membrane) low protein-binding filter (Acrodisc Syringe
Filters, Pall Corp, USA) into sterile test tubes. The sterile SWS was
then stored at -20°C. Prior to use, the SWS was thawed and centrifuged
once again to remove any precipitate.
Preparation of Bacterial Suspension
The stock culture of Strep. mutans was thawed and then inoculated
into 30 mL of sterilized BHI broth (1:100 v/v) accordingly to form the
bacterial suspension. The concentration of the bacteria used in the study
was adjusted using a spectrophotometer to give an absorbance of 0.144
at 550 nm wavelength. At this absorbance, the bacterial cell number is
108 cells mL-1 (Fathilah and Rahim, 2003). This
procedure is important for the standardization of bacterial cell number
in the suspension to be used in the study.
Preparation of Brain Heart Infusion (BHI) Broth Supplemented with
1% Sucrose
The preparation of BHI broth supplemented with 1% sucrose was similar
to the preparation of BHI broth except for the addition of 1% sucrose
to its total volume. The mixture was then sterilized by autoclaving and
kept at 4°C until further use. This media was used as a reservoir
for the bacteria.
Preparation of Brain Heart Infusion Agar (BHI Agar) Media
BHI agar plates were prepared according to the manufacturer`s instruction
(Oxoid Ltd.). The agar plates were stored in the fridge at 4°C until
further use. These plates were used for the enumeration of the colony
forming unit (CFU) of the cultured bacteria.
Experimental Procedure for the Development of Oral Biofilm
In the development of the biofilm, six glass beads were first coated
with saliva by allowing the sterilized saliva to flow into the NAM model
for two minutes at a rate of 0.3 mL min-1. Following that,
sterile distilled water was allowed to run into the system to rinse excess
saliva from the glass beads. The glass beads covered by saliva represent
the acquired pellicle. Bacterial inoculum (Strep. mutans) in the
bacterial reservoir was then pumped into the system at a rate of 0.3 mL
min-1 for 24 h to develop a 24 h-biofilm of Strep. mutans.
For blank control, bacteria-free inoculum was used. The experiment was repeated two times.
Analysis of the Oral Biofilm Formed in the NAM Model
The glass beads with the 24 h biofilm were analyzed for the number
of bacterial cells attached to it. The glass beads located in the middle
(M) and those nearest to the inlet (I) and outlet (O) tubing were chosen
and analyzed for bacterial counts. The glass bead was individually placed
in a test-tube containing Phosphate-Buffered Saline (PBS), sonicated for
a few seconds and vortexed for one minute to dislodge the attached bacteria.
The tube containing the dislodged bacteria was labeled as Tube 1 (T1)
which contained undiluted bacterial count. Tube 1 was then diluted serially
six times (1:10) using the phosphate buffer with the final dilution (6th
dilutions) contained in Tube 7 (T7) (dilution factor = 10-6).
One hundred microliter of the bacterial suspensions from each of the tubes
was drawn and plated on three separate BHI agar plates. These will give
in total 21 BHI agar plates to be incubated aerobically at 37°C for
24 h. The counting of viable microorganisms was carried out on the following
day. Plates from dilution which gave a cfu number of 30-300 cells were
selected. The growth population cfu mL-1 were calculated using
the formula:
CFU count per plate x dilution factor
The result was expressed as mean of cfu count of three plates.
Validation of the Efficiency of the NAM Model in Maintaining the Preset
Conditions
The NAM model was validated for its efficiency in maintaining temperature,
flow rate and sterility throughout the experiment. For temperature, the
water bath was preset at 37°C to mimic the human oral cavity temperature.
It was constantly monitored by the thermometer and recorded every 2 h
for 6 h and this was repeated for three different days. For flow rate,
it was preset at 0.3 mL min-1 to represent the flow rate of
saliva in the oral cavity under unstimulated condition (Edgar and O`Mullane,
2004). It was monitored and recorded every 2 h for 6 h and repeated for
three different days. For sterility, the sterility of the apparatus was
determined in three different days.
Statistical Analysis
Analysis of variance (ANOVA) was used in the statistical analysis.
A p-value of less than 0.05 is considered significant. Comparisons of
the bacterial counts were carried out between the beads within an experiment
and between experiments.
RESULTS AND DISCUSSION
Figure 1 represents the NAM model used in the study. It was found that the bacterial population (cfu mL-1) in the biofilm formed on the glass beads (M, I and O) in each experiment were not
| Table 1: | The bacterial population present in the biofilm formed on each bead was determined and analyzed statistically using ANOVA |
| The bacterial population is expressed as the mean ± standard deviation of three determinations | |
| Table 2: | Comparison of the initial and final readings of the parameters used in the study |
| Fig. 1: | NAM model (A) inside the water bath (incubator) (B) connected to a reservoir (C) via a peristaltic pump. Inset is the enlarged picture of A (indicated by the arrow) |
statistically different (p>0.6) from one another. The standard deviations between experiments 1 and 2 were found to be within 4-5 % (Table 1). The sterility of and temperature and flow rate set for the NAM model prior to the experiment was found to be maintained throughout the experiment (Table 2).
Attempts to simulate and study oral biofilms formation in vitro are not new. Since 1952, many researchers (Pigman et al., 1952; Pigman and Newburn, 1962; Russell and Coulter, 1975; Sudo, 1977; De Jong and Van Der Hoeven, 1987; Busscher et al., 1992; Kinniment et al., 1996; Pratten et al., 1998; Pratten and Wilson, 1999; Weiger et al., 1999; Pratten et al., 2000; Palmer et al., 2001; Hope and Wilson, 2003; Decker et al., 2003; Black et al., 2004; Foster and Kolenbrander, 2004; Lamfon et al., 2005; Steinberg et al., 2005) have already conceived such experiments. Many of the so-called artificial mouth models have used a single chamber containing an enamel surface upon which nutrient or saliva was allowed to flow or drip. The chamber containing the enamel is inoculated directly with oral bacteria and the supply of nutrient allows for colonization of the surface and development of an experimental biofilm. All of these models were designed with the recognition that the flow of saliva and nutrient over the surface are important factors for the development of an oral biofilm.
In this study, the NAM model used a glass chamber to mimic the oral cavity and the glass beads to serve as substrata for biofilm development. The temperature was fixed at 37°C to mimic the in vivo temperature of the mouth and maintained throughout the experiment by immersing the chamber in a water bath which functions as an incubator. A thermometer placed in the incubator kept the temperature of the system in check. This was to ensure that the biofilm develops throughout the experiment in an environment that mimics that of the mouth. Before the bacterial inoculum was pumped into the system, the beads were first coated with saliva to allow for a layer of acquired pellicle or experimental pellicle to form. The flow rate of fluids going in and leaving the chamber was controlled by the peristaltic pump.
The NAM model differs from the previously published models in three aspects. Firstly, it employs a continuous culture system (bacterial reservoir). The bacterial reservoir which contains a population of oral microorganisms in a broth-containing medium supplemented with sucrose is supplied continuously to the biofilm-forming surfaces (glass beads). The top and bottom part of the water bath and reservoir are kept tight with screw-clamps. This ensures that the environment of the bacterial reservoir and the water bath is sterile throughout the experiment. Secondly, it allows for variability of the environment of the bacterial reservoir. Extra nutrient can be added in order to maximize the formation of oral biofilms without affecting the influx of bacteria into the system. The environment can also be varied to suit the requirement for the study. Thirdly, it requires a small space in the laboratory (60 cm (long) x 43 cm (wide)).
The NAM model is valid for use in the development of biofilm as the results obtained on the study are reproducible. This is indicated by the bacterial population (cfu mL-1) of the biofilm formed on the glass beads (M, O and I) which was not significantly different to one another (p>0.6). The standard deviations between the two experiments are within 4-5% implying its usage with good reproducibility.
CONCLUSION
The NAM model can be used in the development of oral biofilms with reproducibility and reliability.
ACKNOWLEDGMENT
This study is supported by Vot F0145/2005A, R and D IRPA No 09-02-03-0197 EA197 and Kuok Foundation Bhd.